Targeted Gene Deletion in Cordyceps militaris Using the Split-Marker Approach

Abstract

The macrofungus Cordyceps militaris contains many kinds of bioactive ingredients that are regulated by functional genes, but the functions of many genes in C. militaris are still unknown. In this study, to improve the frequency of homologous integration, a genetic transformation system based on a split-marker approach was developed for the first time in C. militaris to knock out a gene encoding a terpenoid synthase (Tns). The linear and split-marker deletion cassettes were constructed and introduced into C. militaris protoplasts by PEG-mediated transformation. The transformation of split-marker fragments resulted in a higher efficiency of targeted gene disruption than the transformation of linear deletion cassettes did. The color phenotype of the Tns gene deletion mutants was different from that of wild-type C. militaris. Moreover, a PEG-mediated protoplast transformation system was established, and stable genetic transformants were obtained. This method of targeted gene deletion represents an important tool for investigating the role of C. militaris genes.

Transcriptome Analysis Reveals the Flexibility of Cordycepin Network in Cordyceps militaris Activated by L-Alanine Addition

“…The gene silencing of TFs could further study their function, but the efficient gene inhibition system has not been established in C. militaris. Disruption (Chen et al, 2018;Lou et al, 2018) of the genes is another option. The deletion of the transcription factor will lead to multiple effects, because TFs may bind to multiple DNA elements of different genes in different space-time stages.…”

Transcriptome Analysis of Cordyceps militaris Reveals Genes Associated With Carotenoid Synthesis and Identification of the Function of the Cmtns Gene

“…In order to study the mechanism of carotenoid biosynthesis in C. militaris , we performed functional identification of the Cmtns gene. In our previous studies, we had knocked out the Cmtns gene by homologous recombination and obtained the target gene mutant Δ Cmtns (Lou et al, 2018). In this study, the Cmtns gene was complemented to the Δ Cmtns mutant by the ATMT method.…”

Efficient CRISPR-Cas9 Gene Disruption System in Edible-Medicinal Mushroom Cordyceps militaris

“…The Agrobacterium tumefaciens -mediated transformation (ATMT) was used as described in a previously study (Zheng Z. et al, 2011 ) with some modification. To identify the resistance marker for detecting the transformants of ATMT, conidia of C. militaris were harvested and inoculated onto PDA with different Basta, G418, phleomycin, hygromycin or nourseothricin concentrations (100, 200, 300, 400, 500, 1,000, 2,000, 3,000, and 4,000 μg ml −1 ; Lou et al, 2018 ). In the process of transformation, A. tumefaciens AGL-1 carrying the vector p390-blpR-cmcas9 was grown in LB or IM (Zheng Z. et al, 2011 with 200 mM acetosyringone; Sigma-Aldrich, MO, USA) medium with kanamycin (50 μg ml −1 ) and carbenicillin (50 μg ml −1 ) on a shaker (180 rpm, 28°C).…”